This invention relates to compositions and methods for the in vitro culture, transformation, and regeneration of plants.
Genetic improvement of various crop species by genetic engineering has sometimes been hindered because techniques for in vitro culture, transformation, and regeneration of model cultivars are less effective with recalcitrant commercial cultivars.
Barley (Hordeum vulgare L.) is one of the world""s most important cereal crops, closely following wheat, rice, and maize in total production. Barley is used worldwide for feed, food, and malting purposes.
The ability to genetically engineer barley to improve its performance and pest-resistance or to enhance alternative uses is of great importance. The practical utility of stable transformation technologies is largely dependent on the availability of efficient methods for generating large numbers of fertile green plants from tissue culture materials. Procedures have been described for generating highly embryogenic barley callus and regenerating green plants (Dale and Dambrogio, 1979; Handel et al., 1985; Thomas and Scott, 1985; Goldenstein and Kronstadt, 1986; Lxc3xcrz and Lxc3x6rz, 1987; Wan and Lemaux, 1994; Hagio et al., 1995; Rahleen, 1996). However, presently available procedures for producing embryogenic callus and regenerating green plants have been of limited utility when used in transformation procedures for commercially important barley genotypes. These procedures have been hampered by a gradual loss of the embryogenic capacity and regenerability of callus tissue and an increase in albino (chlorophyll-deficient) plants during the prolonged periods needed to select transformed tissue. For example, of the independently transformed callus lines generated by one transformation procedure for the barley genotype Golden Promise, only 51% of transformed lines give rise to green plants and some of these lines regenerated only a small number of green plants (Wan and Lemaux, 1994; Lemaux et al., 1996). When the same procedure was applied to the commercial barley genotypes Moravian III and Galena, none of the resulting transformed lines gave rise to green plants.
There is a need, therefore, for efficient methods for transformation and regeneration that can be used with a wide variety of barley genotypes, including commercially important genotypes.
We have developed improved methods and compositions for plant transformation and regeneration. The examples below detail the application of these methods and compositions to various barley genotypes, including commercially important genotypes that have proven difficult or impossible to transform and regenerate by previously available methods. These improved methods, when applied to barley, result in a significantly higher regeneration frequency, reduce somaclonal variation, and improve the incidence of fertile, green transformed plants. The methods of the present invention are not limited to barley, however, but can be used for transformation and regeneration of other plant species.
One aspect of the present invention encompasses methods for producing a transformed plant that include an intermediate incubation step that improves the frequency with which transformed plants are obtained from independent transformation events. More specifically, such methods comprise the steps of:
(1) transforming a cell of a target plant tissue (e.g., immature embryo, callus, microspore-derived embryo, etc.) to produce a transformed cell;
(2) culturing the transformed cell on a callus-induction medium (CIM) that includes an auxin to promote proliferation of the transformed cell and formation of a transformed callus, i.e., a callus arising from the initial transformation event (in some embodiments, the CIM also contains a low level of a cytokinin and a high level of copper);
(3) culturing the transformed callus on an intermediate-incubation medium (IIM) that includes an auxin and a cytokinin to promote continued proliferation of cells arising from the initial transformation event and formation of a regenerative structure, i.e., a multicellular structure that is competent to regenerate; and
(4) culturing the regenerative structure on a regeneration medium (RM; i.e., shooting and/or rooting medium) to produce a transformed plant.
Selection for transformed cells can begin immediately after introduction of DNA into a cell. Alternatively, selection can begin later, e.g., during callus induction in order to provide sufficient time for initial cell proliferation in the absence of the selective agent. Selection is generally maintained during the intermediate incubation step and, depending on the selective agent, can also be maintained during the regeneration step.
Another aspect of the present invention encompasses optimized plant culture media and the use of such media for plant cell and tissue culture. Such optimized media include phytohormones and copper (e.g., cupric sulfate), which improve callus quality during initiation, promote the regenerability of the tissue, and reduce the incidence of albinism during the period of callus maintenance and regeneration. The media also includes conventional plant nutrients and can also include a carbon source such as maltose (which is better than sucrose for initiation of some species, including barley, wheat, and rice).
In preferred embodiments, the CIM includes an auxin (e.g., 2,4-dichlorophenoxyacetic acid or dicamba), for example at a concentration of about 0.1 mg/L to about. 5.0 mg/L, preferably about 1.0 mg/L to about 2.5 mg/L. The CIM can also include a cytokinin (e.g., 6-benzylaminopurine, zeatin, and kinetin), e.g., at a concentration of about 0.01 mg/L to about 0.5 mg/L for initial callus induction and about 0.1 mg/L to about 2.0 mg/L for maintenance of callus and green tissues.
In preferred embodiments, the IIM contains an auxin, e.g., at a concentration of about 0.1 mg/L to about 5.0 mg/L, preferably about 0.5 mg/L to about 2.5 mg/L, and a cytokinin, e.g., at a concentration of about 0.1 mg/L to about 5.0 mg/L, preferably about 0.1 mg/L to about 2.0 mg/L.
The CIM and IIM also preferably include copper, e.g., a concentration of about 0.1 xcexcM to about 50 xcexcM.
Another aspect of the present invention encompasses the use of dim light conditions during early phases of selection. Dim light conditions allow callus to become green and reduce the incidence of regeneration of fertile green plants, and may improve the regenerability of the callus tissue. Dim light conditions also permit one to screen for green portions of the callus (for barley, for example; yellow-green portions for wheat), which are more likely to be regenerable. Green callus is useful as a target plant tissue for transformation, e.g., by microprojectile bombardment or infection by Agrobacterium. Callus grown in dim light on a CIM develops or maintains regenerative structures and can be maintained in this state for at least ten months for Golden Promise, Galena, and Harrington, and at least four to six months for Morex, for example.
Another aspect of the present invention is the use of microprojectile bombardment for plant transformation, wherein the bombardment is performed below 1300 psi, e.g. at 450-900 psi. Lowering the rupture pressure and -hence the speed of the microprojectiles lessens damage to the target tissue and results in less stress to the transformed cells.
Another aspect of the present invention encompasses transformed plants and plant culture media as described herein.
The foregoing and other aspects of the invention will become more apparent from the following detailed description.